Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.


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March 2021

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Data exclusions
Replication Randomization Blinding Bioluminescence images were analysed with Living Image software 4.5 (Caliper Life Sciences). Immunofluorescence images analysed with Zen Blue 3.4 (Zeiss). Immunohistochemistry images were analysed with NDP.view2 (Hamamatsu). For scBS-seq analysis, read alignment, deduplication and methylation calling was performed using Bismark v0.22.1. For scRNA-seq analysis, reads were mapped with hisat2 v2. Sample sizes were set to ensure reproducible results. For reporter line validation, at least 3 independent litters were generated for each embryonic and adult time-point, which was sufficient to ensure observations were reproducible. For generational studies and production of F1 and F2 animals, a minimum of 6 female mice were set-up from the F0 and F1 generations, allowing the phenotype to be observed, while also ensuring that excess animals were not used (NC3Rs). For observations of the F3 generation, 3 female mice and 2 male mice were separately set up, providing sufficient F3 offspring to determine that no bioluminescent phenotype could be identified. For QRT-PCR analysis, RNA was extracted from tissues from 4 individuals, providing sufficient numbers for statistical comparisons between groups. Staining was performed on a minimum of 2 individuals, ensuring observations were reproducible; and at least 4 F2-matHFD individuals to observe variability. Bisulphite analysis was performed on two individuals, providing sufficient clones for statistical comparison and calculation of percentage methylation, and ensuring that observations were reproducible between individuals. For the single cell analysis, oocytes from 6 animals per group were taken, producing sufficient oocytes to produce allow robust statistical analysis. For the small RNA analysis, oocytes from 4 animals per group were taken, allowing robust statistical analysis between groups.
15 individual oocyte data sets were excluded, as these failed QC, determined by inappropriate global and/or X-chromosome methylation levels. No other data were excluded.
For reporter line validation, at least 3 independent litters were generated for each embryonic and adult time-point. For generational studies and production of F1 and F2 animals, a minimum of 6 female mice were set-up from the F0 and F1 generations. For observations of the F3 generation, 3 female mice and 2 male mice were separately set up. Staining experiments were performed on a minimum of 2 individuals, and staining was performed in duplicate for each individual. For QRT-PCR each well was pipetted in technical triplicate and each plate run in technical duplicate, with four samples per group. Bisulphite analysis was repeated twice. For the single cell analysis, oocytes from 6 animals per group were taken, producing a total of 41 F1mat-CD and 37 F1mat-HFD informative datasets. For the small RNA analysis, libraries were generated from oocytes taken from 4 animals per group. All replicate attempts were successful, apart from the exclusion of 15 single oocyte datasets as indicated above, and one F1mat-HFD female set up with a wtCD male did not produce any F2 litters (reported in the manuscript).
Female mice were randomly assigned to experimental (dietary) group. No other experiments involved allocation to experimental groups.
Researchers were blinded to groups during data acquisition, including in vivo imaging and molecular biology. Researchers were not blinded during analysis since the data were analysed according to defined comparisons.